Download Stimulation of Cytokine Expression by Peripheral Blood

NEONATOLOGY
Stimulation of Cytokine Expression by Peripheral
Blood Mononuclear Cells of Neonatal Foals Using
CpGs
Tong Liu, MS, PhD; Noah Cohen, VMD, MPH, PhD, Diplomate ACVIM*;
Jessica Nerren, PhD; Jennifer Murrell, MS; Veronique Juillard, PhD; and
Ronald Martens, DVM
Young foals are particularly susceptible to infectious diseases. Cytosine-phosphate-guanosine dinucleotide oligodeoxynucleotides can stimulate cytokine expression by peripheral blood mononuclear
cells of young foals. This stimulation of innate immunity might aid in protecting foals against
infectious diseases. Authors’ addresses: Equine Infectious Disease Laboratory, Department of
Large Animal Clinical Sciences, College of Veterinary Medicine, Texas A&M University, College
Station, TX 77843-4475 (Liu, Cohen, Nerren, Murrell, Martens); and Merial SAS Discovery, Lyon,
France (Juillard); e-mail: [email protected] (Cohen). © 2008 AAEP. *Presenting author.
1.
Introduction
Neonatal foals are susceptible to a variety of microbial infections that are generally associated with
underlying immunodeficiencies in people and other
animals, such as Candida albicans, Cryptosporidium parvum, Pneumocystis carinii, and Rhodococcus equi. These infections are rarer in adult
horses than foals, suggesting an age-related deficiency in immunity. Innate and adaptive immune
responses are diminished in human neonates, and
these deficiencies predispose to infectious disease.1
Innate immune responses provide the first line of defense against infections and serve to instruct the development of adaptive immune responses.1 There is
evidence that interferon ␥ (IFN␥), an important mediator of innate immune responses, is relatively deficient
in neonatal foals, and it has been proposed that this
deficiency biases foals away from a T helper 1 (Th1)type response, thus rendering foals more susceptible to
infection.2,3 Therefore, enhancing expression of IFN␥
and other pro-inflammatory cytokines might help protect foals against infection. Cytosine-phosphateguanosine (CpG) dinucleotide motifs found frequently
in bacterial DNA induce a strong Th1-type immune
response, and synthetic CpG oligodeoxynucleotides
(CpG-ODNs) are used as adjuvants against bacterial
infectious diseases, neoplasms, and allergic disorders.4 – 6 The objective of this study was to investigate the extent to which peripheral blood mononuclear
cells (PBMCs) from foals could be stimulated by selected proprietary CpG-ODNs to express mRNA of the
pro-inflammatory cytokines IFN␥, tumor necrosis factor ␣ (TNF␣), and interleukin (IL)-6, IL-8, IL-12p35,
and IL-12p40 and to determine the extent to which
fold changes in expression of mRNA for these cytokines differed between mares and foals.
2.
Materials and Methods
PBMCs were collected from fresh, heparinized, venous blood from five healthy Quarter Horse foals at
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NEONATOLOGY
14 and 56 days of age and from their dams when the
foals were ⬃21 days old. The PBMCs were divided
into six aliquots such that one aliquot of 1 ⫻ 106 was
incubated with each of the four CpG-ODNs (50 ␮g/
ml) for 24 h and one aliquot of 1 ⫻ 106 was incubated
for 23 h in media only, followed by 1 h of incubation
with a virulent isolate (i.e., an isolate bearing the
80- to 90-kb virulence-associated plasmid) of R. equi
(strain ATCC 33701; 1 ⫻ 107 cells/well) as a positive
control for expression of IFN␥, TNF␣, and IL-6. The
remaining aliquot of PBMCs was incubated with
only media for 24 h as a negative control. After
incubation, total RNA was extracted from the cells,
reverse transcribed, and subjected to real-time polymerase chain reaction (PCR) to determine levels of
expression of IFN␥, TNF␣, IL-6, IL-8, IL-12p35, and
IL-12p40.
Expression of each cytokine relative to baseline
(i.e., cells incubated in media only for 24 h) was
calculated for each CpG-ODN using the ⌬⌬Ct
method. To account for the repeated measurements on individual foals, mixed-effects models
were used for analysis of data, with foal modeled as
a random effect, and age, R. equi, and CpG type as
fixed effects. Age was modeled as a continuous
variable, and the reference for stimulatory CpGODNs was CpG 2041 (referred to as the control
ODN). It was also possible to make inferences
about differences relative to the unstimulated control by evaluating the intercept term for each model.
A similar modeling approach was used for the mare
data, except that age was not considered as a factor.
For a given cytokine, an ANOVA was performed
comparing the values of differences between mares
and foals (using a model for the paired differences at
14 days and another model for the paired differences
at 56 days) by CpG; post-hoc testing of differences
among the various CpGs was performed using
Tukey’s least significant difference method. A significance level of p ⬍ 0.05 was used for analysis.
3.
Results
Relative to PBMCs that were unstimulated or stimulated with the control CpG-ODN (CpG 2041), each
of the stimulatory CpG-ODNs induced significant
(p ⬍ 0.05) mRNA of IFN␥, IL-6, and IL-12p40 from
PBMCs from foals at 14 and 56 days and from
mares. There was no significant induction of expression of TNF␣, IL-8, or IL-12p35 by the CpGODNs relative to the control CpG-ODN in mares or
foals. There were no differences in expression levels observed between foals that were 14 and 56 days
of age, and there were no significant differences
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between mares and foals in fold changes in expression of any of the cytokines. The mRNA expressions of IFN␥, IL-6, and IL-12p40 stimulated by
CpG-ODNs 2135 and 2142 were ⬃2-fold higher than
those induced by CpG-ODN 2395, although the differences were significant only for IL-12p40 and IL-6.
4.
Discussion
Because foals may become infected with a variety of
pathogenic microbes early in life at a time when
their immune system is relatively immature and
before the time in which an effective adaptive immune response can be developed for protection,
strategies to stimulate innate immune responses
might provide effective protection for foals against
infectious diseases. This protection might result
both directly by enhancing innate responses and
indirectly from the critical role that innate immunity plays in development of adaptive immune responses. Foals produce relatively less IFN␥, IL-6,
and other cytokines shortly after birth.2,3,7 Results
of this study indicate that CpG-ODNs can effectively
stimulate mRNA expression of IFN␥, IL-6, and IL12p40 by PBMCs of foals as young as 2 wk of age.
Moreover, the magnitude of the relative fold change
in expression of IFN␥ by foals was at least as strong
if not stronger than that observed in mares. These
findings indicate that CpG-ODNs might be useful as
immunomodulators or as potential adjuvants for
vaccines to aid in preventing R. equi pneumonia and
other bacterial diseases of neonatal foals.
This work was supported by Merial, with additional funding from the Link Equine Research Endowment, Texas A&M University.
References
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